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The bromodomain and extra-C-terminal domain (BET) protein family is a class of epigenetic readers that recognize acetyl-lysine residues of histones, and their targeting with small molecules is a no...

The reaction between fission products Mo(VI) and Zr(IV) in hot nitric acid solution during the spent fuel dissolution process leads to the formation of hydrated zirconium molybdate precipitate. Thi...

// Maria E. Riveiro 1 , Lucile Astorgues-Xerri 1 , Ramiro Vazquez 2 , Roberta Frapolli 2 , Ivo Kwee 3, 5 , Andrea Rinaldi 3 , Elodie Odore 6 , Keyvan Rezai 6 , Mohamed Bekradda 1 , Giorgio Inghirami 7, 9 , Maurizio D’Incalci 2 , Kay Noel 10 , Esteban Cvitkovic 1, 10 , Eric Raymond 11 , Francesco Bertoni 3, 12 1 Oncology Therapeutic Development, Clichy, France 2 IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 3 Lymphoma and Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, Switzerland 4 Dalle Molle Institute for Artificial Intelligence (IDSIA), Manno, Switzerland 5 SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland 6 Radiopharmacology Department, Curie Institute - Rene Huguenin Hospital, Saint Cloud, France 7 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA 8 Department of Molecular Biotechnology and Health Science and Center for Experimental Research and Medical Studies (CeRMS), University of Turin, Turin, Italy 9 Department of Pathology, and NYU Cancer Center, New York University School of Medicine, New York, NY, USA 10 Oncoethix SA (now Oncoethix GmbH, a wholly owned subsidiary of Merck Sharp and Dohme Corp.), Lucerne, Switzerland 11 Medical Oncology Department, CHUV, Lausanne, Switzerland 12 Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland Correspondence to: Maria E Riveiro, email: eugenia.riveiro@oncotd.com Keywords: OTX015 (MK-8628), bromodomain, NSCLC, SCLC, KRAS Received: January 21, 2016 Accepted: October 14, 2016 Published: November 07, 2016 ABSTRACT Inhibitors targeting epigenetic control points of oncogenes offer a potential mean of blocking tumor progression in small cell and non-small cell lung carcinomas (SCLC, NSCLC). OTX015 (MK-8628) is a BET inhibitor selectively blocking BRD2/3/4. OTX015 was evaluated in a panel of NSCLC or SCLC models harboring different oncogenic mutations. Cell proliferation inhibition and cell cycle arrest were seen in sensitive NSCLC cells. MYC and MYCN were downregulated at both the mRNA and protein levels. In addition, OTX015-treatment significantly downregulated various stemness cell markers, including NANOG, Musashi-1, CD113 and EpCAM in H3122-tumors in vivo . Conversely, in SCLC models, weak antitumor activity was observed with OTX015, both in vitro and in vivo . No predictive biomarkers of OTX015 activity were identified in a large panel of candidate genes known to be affected by BET inhibition. In NSCLC models, OTX015 was equally active in both EML4-ALK positive and negative cell lines, whereas in SCLC models the presence of functional RB1 protein, which controls cell progression at G1, may be related to the final biological outcome of OTX015. Gene expression profiling in NSCLC and SCLC cell lines showed that OTX015 affects important genes and pathways with a very high overlapping between both sensitive and resistant cell lines. These data support the rationale for the OTX015 Phase Ib (NCT02259114) in solid tumors, where NSCLC patients with rearranged ALK gene or KRAS-positive mutations are currently being treated.

// Eugenio Gaudio 1 , Chiara Tarantelli 1 , Maurilio Ponzoni 2 , Elodie Odore 3 , Keyvan Rezai 3 , Elena Bernasconi 1 , Luciano Cascione 1, 4 , Andrea Rinaldi 1 , Anastasios Stathis 4 , Eugenia Riveiro 5 , Esteban Cvitkovic 5 , Emanuele Zucca 4 , Francesco Bertoni 1, 4 1 Lymphoma and Genomics Research Program, Institute of Oncology Research (IOR), Bellinzona, Switzerland 2 San Raffaele Scientific Institute, Milan, Italy 3 Institut Curie, Hopital Rene Huguenin, Saint-Cloud, France 4 Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland 5 Oncology Therapeutic Development, Clichy, France Correspondence to: Francesco Bertoni, email: frbertoni@mac.com Keywords: BET inhibitor, ibrutinib, rituximab, vorinostat, everolimus Received: April 28, 2016 Accepted: July 07, 2016 Published: August 1, 2016 ABSTRACT The bromodomain inhibitor OTX015 (MK-8628) has shown anti-lymphoma activity as a single agent in both the preclinical and clinical settings, as well as in vitro synergism with several anticancer agents. Here, we report in vivo data for OTX015 in combination with the histone deacetylase inhibitor vorinostat, the Bruton’s tyrosine kinase inhibitor ibrutinib, the anti-CD20 monoclonal antibody rituximab, and the mTOR inhibitor everolimus in a diffuse large B cell lymphoma model. The antitumor effect of OTX015-containing combinations in SU-DHL-2 xenografts in mice was much stronger than the activity of the corresponding single agents with almost complete tumor eradication for all four combinations. Pharmacokinetic analyses showed similar OTX015 levels in plasma and tumor samples of approximately 1.5 μM, which is equivalent to the concentration showing strong in vitro activity. For all four combinations, mean terminal levels of the bromodomain inhibitor differed from those in mice exposed to single agent OTX015, indicating a need for thorough pharmacokinetic investigations in phase I combination studies. In conclusion, our results provide a strong rationale to explore OTX015-containing combinations in the clinical lymphoma setting.

Bromodomain and extraterminal (BET) bromodomain (BRD) proteins are epigenetic readers that bind to acetylated lysine residues on chromatin, acting as co-activators or co-repressors of gene expression. BRD2 and BRD4, members of the BET family, are significantly increased in glioblastoma multiforme (GBM), the most common primary adult brain cancer. OTX015 (MK-8628), a novel BRD2/3/4 inhibitor, is under evaluation in dose-finding studies in solid tumors, including GBM. We investigated the pharmacologic characteristics of OTX015 as a single agent and combined with targeted therapy or conventional chemotherapies in glioblastoma cell lines. OTX015 displayed higher antiproliferative effects compared to its analog JQ1, with GI50 values of approximately 0.2 µM. In addition, C-MYC and CDKN1A mRNA levels increased transiently after 4 h-exposure to OTX015, while BRD2, SESN3, HEXIM-1, HIST2H2BE, and HIST1H2BK were rapidly upregulated and sustained after 24 h. Studies in three additional GBM cell lines supported the antiproliferative effects of OTX015. In U87MG cells, OTX015 showed synergistic to additive activity when administered concomitant to or before SN38, temozolomide or everolimus. Single agent oral OTX015 significantly increased survival in mice bearing orthotopic or heterotopic U87MG xenografts. OTX015 combined simultaneously with temozolomide improved mice survival over either single agent. The passage of OTX015 across the blood-brain barrier was demonstrated with OTX015 tumor levels 7 to 15-fold higher than in normal tissues, along with preferential binding of OTX015 to tumor tissue. The significant antitumor effects seen with OTX015 in GBM xenograft models highlight its therapeutic potential in GBM patients, alone or combined with conventional chemotherapies.

OTX015 (MK-8628) is a novel inhibitor of the bromodomain and extraterminal (BET)-bromodomain (BRD) protein family, binding specifically to bromodomains BRD2/3/4 and impacting the epigenetic regulation of several oncogenes. We characterized the pharmacokinetics of this first-in-class BET-BRD inhibitor administered as a single agent, including population pharmacokinetic modelling. A dose-escalation, phase Ib study was performed with oral OTX015 in patients with haematologic malignancies, at doses starting from 10 mg once daily (QD) with continuous or discontinuous schedules. Five or eight blood samples were collected per patient for pharmacokinetic analysis. OTX015 plasma concentrations were determined using validated ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) and analysed using a nonlinear mixed-effects modelling software program. A population pharmacokinetic model was fitted to the data, and patient demographics and clinical chemistry parameters were tested as predictive covariates on the model parameters. Blood samples were analysed from 81 patients treated with OTX015 at doses ranging from 10 to 160 mg QD or 40 mg twice daily (BID), and 633 time–plasma concentrations were available for analysis. A one-compartment open model with linear elimination adequately described OTX015 pharmacokinetics. The most significant covariate was lean body mass (LBM), which decreased the between-subject variability in apparent total body clearance (CL) and the volume of distribution (V). The estimated pharmacokinetic parameters were the absorption rate constant (k a) = 0.731 h−1, V = 71.4 L and CL = 8.47 L·h−1. The pharmacokinetics of oral OTX015 in patients with haematologic malignancies can be described with a one-compartment model. Population pharmacokinetic modelling of OTX015 plasma concentrations showed that LBM influences V and CL. These findings do not suggest the need for dose adjustment.

OTX015 is a novel synthetic thienodiazepine analog, which potently inhibits bromodomains (BRD) 2, 3 and 4 of the BET (bromodomain and extraterminal) protein family. It is currently undergoing phase I evaluation in patients with hematologic malignancies using an oral formulation. We developed and validated an Ultra Performance Liquid Chromatography method with tandem Mass Spectrometry detection (UPLC-MS/MS) for quantification of OTX015 in plasma in order to investigate its pharmacokinetics in humans, using small plasma samples (50 μL), and an internal standard, Y-401. Chromatographic separation was performed on a BEH C18 UPLC column with a mobile phase gradient at a flow rate of 0.5 mL min−1 for 5 minutes. Quantification was performed using the transition 492–383 (m/z) for OTX015 and 506–383 (m/z) for Y-401. The lower limit of quantification (LLOQ) was established as 1 ng mL−1 with 10.80% precision and 94.67% accuracy. The calibration curve was linear up to 250 ng mL−1 (upper limit of quantification). Intra-assay precision ranged from 7.3% to 11.6% for the three quality control (QC) concentrations evaluated and was 16.0% for the LLOQ, and intra-assay accuracy ranged from 93.7% to 109.8% for the three QC concentrations and was 92.0% for the LLOQ. Inter-assay precision and accuracy ranged from 4.1% to 14.0% and from 92.3% to 104.8%, respectively. These data show that the UPLC-MS/MS procedure is sensitive, accurate, precise and robust, and was validated for determining OTX015 concentrations in plasma. The method was successfully applied to determine the pharmacokinetic profile of OTX015 in patients treated in an ongoing phase I clinical study.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75-650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).

Background: BRD2 and BRD4, members of the BET family of bromodomains, were recently described to be significantly increased in glioblastoma multiforme (GBM), the most common primary adult brain cancer. OTX015 (OncoEthix SA, Switzerland), a novel BET-BRD2/3/4 inhibitor, is currently being evaluated in clinical Phase Ib studies in hematologic malignancies and solid tumors, including GBM. Here, we report the effects of OTX015 in vitro as a single agent and in combination with temozolomide (TMZ) in human U87MG cells, along with in vivo activity in heterotopic and orthotopic U87MG models. Material and Methods: GI50s were determined in U87MG cells after 72 h with OTX015 or JQ1 with MTT assays. Combination indexes of simultaneous and sequential schedules of OTX015 with TMZ were determined using Chou & Talalay analysis. For orthotopic in vivo experiments, 105 U87MG cells were injected in the frontal lobe of nude mice and 5 days later mice were randomized. For the xenograft model, 5.106 U87MG cells were injected into the right lateral flank and mice were randomized when tumors reached 100 mm3. Treatment groups (n = 6) were: vehicle PBS, OTX015 50 mg/kg/BID and 100 mg/kg/daily, or TMZ 100 mg/kg/daily (ip, days 6-10). Median survival was determined in the orthotopic model by the Kaplan-Meier method. Tumor volume was evaluated 3 times weekly in the flank model. An independent in vivo experiment was done for the evaluation of OTX015 levels in tumor and normal tissues, animals were sacrificed after 7 days of treatment at 50 mg/kg/BID, using ultra performance liquid chromatography with tandem mass spectrometry. Results: OTX015 displayed antiproliferative effects in U87MG cells with GI50 values of 0.9μM versus 2.1μM for JQ1. OTX015/TMZ combination studies revealed synergistic activity when TMZ was administered after OTX015. OTX015 significantly increased survival in mice bearing orthotopic U87MG cells, with median survival of 28 and 25 days for 50 and 100 mg/kg/daily OTX015, respectively vs 19.5 days in control mice. Furthermore, in the heterotopic model, OTX015 treatment at both doses, significantly decreased tumor growth at day 22. No major side effects were seen during in vivo OTX015 treatment. In orthotopic models, OTX015 plasmatic levels were 1365.3 (±481)ng/mL, while in tumor and peritumoral brain tissues OTX015 levels were 995.0(±549) and 62.97(±10.3)ng/g tissue, respectively. In the flank model, OTX015 tumor levels were about 1.2 times higher than in peripheral muscle tissue and plasma levels [848.1(±127)ng/mL] and about 7 times higher than in normal brain tissue. Conclusion: We demonstrated the passage of OTX015 across the blood-brain barrier, as well as preferential binding of OTX015 to tumor tissue. Oral OTX015 treatment significantly enhanced survival and decreased tumor growth in GBM xenograft models, highlighting the therapeutic potential of OTX015 in GBM patients. Citation Format: Lucile Astorgues-xerri, Caroline Berenger, Mylène Cayol, Mohamed Bekradda, Elodie Odore, Keyvan Rezai, Esteban Cvitkovic, Maria E. Riveiro, L'Houcine Ouafik. OTX015, a novel BET-bromodomain (BET-BRD) inhibitor, displays antitumoral effects in orthotopic and heterotopic models of human glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3527. doi:10.1158/1538-7445.AM2015-3527

Background: OTX015 (OncoEthix SA, Switzerland) is a novel oral bromodomain and extraterminal (BET) protein family inhibitor, with in vitro and in vivo activity in a variety of hematologic and solid tumor cells. A phase Ib study with OTX015 in patients with hematologic malignancies is underway including a pharmacokinetic (PK) investigation. The PK objectives of this study were to determine the PK profile of oral OTX015 using a population approach. Materials and Methods: A multicenter, dose escalation study in cohorts of 3 to 6 patients with acute leukemia or other hematologic malignancies was performed with a dose escalation step followed by expansion cohorts at the recommended dose. Patients received oral OTX015 from 10 to 160 mg different schedules. PK blood samples from 7 time points were collected over 24 h post-administration on Day 1 for leukemia patients (complete PK) and 4 blood samples over 8 h post-administration for patients with other hematologic malignancies (limited PK). OTX015 plasma concentrations were measured using validated ultra-performance liquid chromatography with tandem mass spectrometry detection with a concentration range 1-250ng/mL. Analyses and population PK (PPK) modeling were performed with the nonlinear mixed effect modeling software program Monolix version 4.3. The following parameters were calculated absorption constant (Ka); apparent distribution volume (V/F); apparent clearance (CL/F) and lean body mass (LBM; calculated considering patient sex, weight and height) was considered as covariate. Results: 85 patients enrolled and treated from January 2013 to August 2014, randomized to six dose levels (10, 20, 40, 80, 120 and 160 mg) QD and 40 mg BID were evaluated. Among them, 81 patients with 630 plasma concentrations (607 + 23 BLQ) were evaluable for PK assessment. A 1-compartment open model adequately described the total OTX015 concentration-time curve. The PPK parameters obtained for the structural model were Ka = 0.74 h−1 (12%); V/F = 71.7 L (6.0%) and CL/F = 8.45 L/h (5.0%). The best correlation between OTX015 AUC values and dose was observed from 10 to 120 mg dose levels (R2 = 0.71). The absorption phase was linear and Tmax was between 1 and 4 h. Mean elimination half-life of OTX015 for all patients was 5.8 h (± 1.1). In the PPK study, the best descriptive model was obtained when LBM was considered in the analysis. A correlation between CL/F and V/F was also observed for OTX015. Conclusions: The PK of OTX015 is best described by a one-compartment model. Preliminary PPK analysis considering only the dose escalation cohort of the Phase I trial indicates that LBM is a good predictor of the OTX015 PK profile. This model should be validated in the ongoing expansion cohorts treated at 80mg QD. Citation Format: Elodie Odore, Francois Lokiec, Maria Eugenia Riveiro, Fabrice Bourdel, Carmen Kahatt, Patrice Herait, Esteban Cvitkovic, Keyvan Rezai. Pharmacokinetics of OTX015 in a phase Ib dose-finding study of patients with hematologic malignancies: Preliminary results of a population PK analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2015-4511

Background: OTX015 is a novel inhibitor of the bromodomain and extra-terminal (BET) protein family. This synthetic oral small molecule targets the BET transcriptional co-activator proteins BRD2/3/4, which are potential cancer targets particularly in hematologic malignancies. A phase Ib study with OTX015 administered to patients with hematologic malignancies was performed including a pharmacokinetic (PK) investigation. The PK objectives of this study were to determine the PK profile and perform a PK/PD modeling of oral OTX015 using a population approach. Materials and Methods: A multicenter, dose escalation study is underway in cohorts of 3 to 6 patients with acute leukemia and other hematologic malignancies. Patients received oral OTX015 at a starting dose of 10 mg once daily (QD). PK blood samples from 7 time points were collected over 24 h post-administration (complete PK) on Day 1 for leukemia patients and 4 blood samples over 8 h post-administration were collected for patients with other malignancies (limited sampling PK). Plasma concentrations of OTX015 were measured using validated Ultra Performance Liquid Chromatography with tandem Mass Spectrometry detection (UPLC-MS/MS) with a concentration range 1 - 250 ng/mL. Analyses and population PK (PPK) modeling were performed with the nonlinear mixed effect modeling software program Monolix version 4.2. Results: From January 2013 to January 2014, 36 patients were treated at four dose levels (10, 20, 40, 80 mg) QD and 40 mg BID. 302 plasma concentrations (289 + 13 BLQ) were analyzed. A 1-compartment open model adequately described the total OTX015 time-concentration curve. The PPK parameters obtained for the structural model were: Ka (absorption constant) = 1.12 h-1; V (distribution volume) = 68.6 L and CL (clearance) = 6.65 L/h with a relative standard error of 27%, 9% and 10% respectively. AUC values for all patients increased dose-proportionally (R²= 0.995). The absorption phase was linear and Tmax was between 1 and 4 hours. Mean elimination half-life of OTX015 for all patients was 7.16 h. The main covariate effects in PPK modeling were body weight (BW) which influenced CL, V. No significant gender influence on PK parameters was observed. Conclusions: The PK of OTX015 is best described by a one-compartment model. BW influenced significantly PK parameters of OTX015. AUC-dose proportionality was observed. Evaluation of glucuronidated metabolite concentrations is ongoing and will contribute to understanding the pathways involved in OTX015 metabolism. PK/PD modeling will be performed to describe toxicity and efficacy (6 experienced clinically meaningful activities) in terms of OTX015 PK. A comparison between QD and BID schemes will be performed in order to verify PK profile of OTX015 for each administration. Citation Format: Elodie Odore, Keyvan Rezai, Eugenia Riveiro, Fabrice Bourdel, Patrice Herait, Esteban Cvitkovic, Herve Dombret, Francois Lokiec. A phase I pharmacokinetic study of OTX015 for the treatment of patients with hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-231. doi:10.1158/1538-7445.AM2014-LB-231

Aim: The bromodomain and extraterminal (BET) subfamily of human bromodomain (BRD) proteins associates with acetylated chromatin and plays a key role in the epigenetic control of transcriptional activation, notably of genes with super-enhancers, such as the MYC oncogene. OTX015, a potent small molecule inhibitor of BRD2/3/4 (Noel et al, EORTC-NCI-AACR 2013), inhibits proliferation of a wide range of hematologic malignancies (HMs) in vitro (Bonetti et al, EORTC-NCI-AACR 2012; Boi et al, EORTC-NCI-AACR 2013; Braun et al, ASH 2013). This phase I study, designed to determine the recommended dose and pharmacokinetics of oral OTX015 as a single agent, is the first reported clinical study evaluating the effects of BRD inhibition in patients (pts) with HMs. Methods: Two independent cohorts of pts having failed all standard therapies were given ascending oral doses of OTX015 in a conventional 3+3 design, with acute leukemias (AL) treated 14 days on/7 days off and other HMs (OHM) treated continuously in 21-day cycles. OTX015 was given once daily (QD), then twice daily (BID). Results: From Jan to Dec 2013, 16 pts with AL (14 AML, 2 ALL) and 17 with OHM (6 DLBCL, 5 other lymphomas, 6 multiple myelomas) were enrolled over 4 dose levels, 10, 20, 40 and 80 mg QD. Exposure increased dose-proportionally. Plasma trough concentrations at 80 mg QD and 12h concentrations at 40 mg QD were ≥ IC50 values in vitro (250 nM), justifying the shift to a BID schedule. The 40 mg BID cohort is ongoing. Pts have a median age of 70 years (range 32-83) and median of 2 (1-8) prior therapies; 10 of 16 AL pts had AML secondary to pre-existing conditions or chemotherapy. No dose limiting toxicity was observed up to 80 mg QD/40 mg BID. Adverse events (AEs) were mainly grade (G) 1-2 hematologic and gastrointestinal events and diabetes aggravation. G 3-4 AEs were reversible thrombocytopenia in 3 pts with OHM (40 and 80 mg), and neutropenia, diarrhea, and elevated transaminases in 1 pt each. No cumulative toxicity was observed. Nine pts received >3 (range 4-7) cycles without or with minor interruptions. Among 28 pts evaluable for response, 6 had clinically meaningful activity, with 4 of 6 treated at 80 mg. Four pts with refractory/relapsed secondary or post-treatment AML achieved significant peripheral and bone marrow blast decrease or clearance, including 1 complete remission (CR) and 1 CR with incomplete recovery. Among OHM, 1 DLBCL had a partial response (PR) and 1 lymphoplasmacytic lymphoma had metabolic PR on cycle 2 PET-scan. Treatment of 5 of 6 responding pts is ongoing. Responses occurred in pts with various clinical, cytogenetic and molecular profiles. Conclusion: OTX015 is the first BRD inhibitor demonstrating clinical activity. Maximum tolerated dose was not reached at 80 mg QD or 40 mg BID; dose escalation is ongoing with the BID schedule and further schedule optimization. Updated results will be presented. Citation Format: Patrice E. Herait, Celine Berthon, Catherine Thieblemont, Emmanuel Raffoux, Valeria Magarotto, Anastasios Stathis, Xavier Thomas, Xavier Leleu, Carlos Gomez-Roca, Elodie Odore, Christophe Roumier, Fabrice Bourdel, Bruno Quesnel, Emanuele Zucca, Mauricette Michallet, Christian Recher, Esteban Cvitkovic, Keyvan Rezai, Claude Preudhomme, Thierry Facon, Antonio Palumbo, Herve Dombret. BET-bromodomain inhibitor OTX015 shows clinically meaningful activity at nontoxic doses: interim results of an ongoing phase I trial in hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT231. doi:10.1158/1538-7445.AM2014-CT231

Background: The human BET family bromodomains which consists of BRD2, BRD3, BRD4 and BRDT proteins has become a druggable target for the development of specific gene transcription inhibitors. Here, we report anti-proliferative activity of OTX015, an orally bioavailable small-molecule BRD-inhibitor which displays high potency and specificity to BRDs 2, 3, and 4, across a large panel of human solid tumor cell lines. Material and Methods: Established 20 human cancer cell lines derived from head and neck, lung, liver, colorectal, renal and pancreatic cancers, were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). MTT assays were performed after 72 hours exposure. GI50 (CI95%) values were estimated using GraphPad Prism 3.0. Cell lines displaying GI50 values500nM were considered insensitive. Genomic DNA from the cell lines was PCR-amplified, sequenced, and assessed for potential sequence alterations (ABI BigDye Terminator Sequencing kit) in the genes KRAS (exon 2 and 3); BRAF (exon 11 and 15); EGFR (exon 20) and PI3KCA (exon 20). Protein levels were analyzed by Western Blot using commercial antibodies. For cell cycle analysis, cells were stained with propidium iodide and analyzed for DNA content using a FACScan flow cytometer. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions. RT- PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. Results: Twenty cell lines derived from a wide range of solid tumor types, belonging to head and neck squamous cell carcinoma (HNSCC), lung adenocarcinoma (LA), hepatocarcinoma (HCC), colorectal carcinoma (CRC), renal (RC), pancreatic (PC) and triple-negative breast cancer (BC) cells were exposed to various concentrations of OTX015 (from 3µM to 9 nM) for 72 h. Eight cell lines displayed GI50 values lower than 500 nM, ranging from 45.9 (15.5-135.1)nM in HT-29 cells (CRC) to 432.0 (38.0-618.7)nM in A-549 (LA) cells. Baseline BRD4/2, c-MYC, BCL-2, CyclinD1 m-RNA leves and protein were characterized in our panel of cell lines and no difference in BRD4/2, c-MYC or BCL-2 basal levels was observed between OTX015 sensitive or resistant cell lines. In addition, no correlation was found between OTX015 anti-proliferative activity and the most prevalent mutations seen in cancer cells (KRAS, BRAF, EGFR and PI3K). In sensitive cell lines, OTX015 caused a cell cycle arrest in G1 in a dose-dependent manner without an increase in cell death (sub-G0 increased). No c-MYC protein level down-regulation was observed after treatment (500nM; 72h) in most of the OTX015 sensitive cell lines, suggesting that alternative pathways can be affected by BRD-inhibition. Noteworthy, after 72 h exposure, significant changes in the cell morphology such as expanded cytoplasm, cytoskeleton reorganization and pyknotic nuclei observed in OTX015 treated cells compared to vehicle treated group, further studies are being conducted to characterize these findings. Conclusion: Together, these findings suggest that OTX015 is active across a wide range of tumor types but this activity could not be restricted only cancers with genomic alterations resulting in MYC overexpression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A121. Citation Format: Maria Eugenia Riveiro, Lucile Astorgues-Xerri, Elodie Odore, Mohamed Bekradda, Esteban Cvitkovic, Kay Noel, Eric Raymond. Preclinical evaluation of OTX015 a novel BET-BRD inhibitor across a panel of solid tumor cell lines of different lineages. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A121.

Background: PDX is a novel 10-deaza-aminopterin analogue of methotrex[[Unsupported Character - Codename ­]]ate consisting of a mixture of R- and S-diastereomeric folate derivatives. PDX inhibits folate metabolism by binding to and inhibiting the enzyme dihydrofolate reductase (DHFR). PDX is the first drug approved by the US Food and Drug Admin[[Unsupported Character - Codename ­]]istration (FDA) specifically for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma. Mass balance studies in animals and humans with radiolabeled compounds represent a standard part of the development process for new drugs. Isotopically labeled analogs of a drug or its metabolites play an important role in understanding the absorption, distribution, metabolism and excretion profiles of a compound. The objectives of this study were to determine the pharmacokinetics (PK), metabolism and routes of excretion of [14C]-PDX and to characterize its metabolites in human plasma and urine. Methods: Four patients were administered intravenously with a mixture of 50 μCi of [14C]-PDX (0.5 mg) and 224.5 mg of non radioactive PDX over a 3-5 minute infusion. Serial blood and plasma samples were drawn at: 0 (before PDX infusion), and up to 144 h after the start of infusion. Urine and fecal samples were collected for up to 168 h after the start of infusion. Expired air samples were collected up to 24 hours after administration. The radioactivity measurement in biological fluids was performed using liquid scintillation counting. Results: Mass balance was achieved with a mean recovery radioactivity in excreta= 82.9 % ± 20.0 % for the four patients. The mean recovery of radioactivity in urine, feces, and expired air were 34.1%, 38.7% and 10.1%, respectively, indicating renal and fecal excretions were the major route of elimination of [14C]-PDX. A three-compartment open model adequately described [14C]-PDX time radioactivity-concentration courses. The AUC0-∞, and clearance values for total radioactivity in plasma were 18.1 nCi.h/mL, and 3.95 L/h, respectively. Metabolite profiling will be performed using HPLC with beta radiation detector. Conclusions: This study quantified the mass balance of PDX and defined its substantial excretion in both urine and feces after IV administration. The PK of [14C]-PDX was best described by a three compartment open model. The metabolite profiling of PDX is ongoing. Citation Format: Elodie Odore, Keyvan Rezaï, Olivier Madar, Sophie Weill, Ahmad Awada, Bert Pronk, Esteban Cvitkovic, Francois Lokiec. Massbalance, excretion and metabolism of [14C]-Pralatrexate (PDX) incancer patients in a phase I trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3367. doi:10.1158/1538-7445.AM2013-3367